A novel subunit vaccine based on Fiber1/2 knob domain provides full protection against fowl adenovirus serotype 4 and induces stronger immune responses than a Fiber2 subunit vaccine.

Outbreaks of hepatitis-hydropericardium syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry in China since 2015. However, commercially available vaccines against the FAdV-4 infection remain scarce. In our study, subunit vaccine candidates derived from the bacterially expressed recombinant Fiber1 knob domain and Fiber2 knob domain fusion protein (termed as Fiber1/2 knob subunit vaccine) and Fiber2 protein (termed as Fiber2 subunit vaccine) of the FAdV-4 SDSX strain were developed. Immunogenicity evaluation showed that the Fiber1/2 knob subunit vaccine induced the production of antibodies at 7 d postvaccination (dpv), earlier than the Fiber2 subunit vaccine. Moreover, the neutralizing antibody level of the Fiber1/2 subunit vaccine group was higher than the Fiber2 subunit vaccine group, showing significant differences at 14, 21, and 28 dpv. Immune protection test results revealed that both Fiber1/2 knob subunit and Fiber2 subunit vaccines could protect chickens from death against FAdV-4 challenge, although the weight of chickens in the Fiber1/2 knob subunit vaccine group decreased less. Furthermore, analysis of plasma Glutamic oxaloacetic transaminase (AST) and blood glutamic pyruvic transaminase (ALT) levels suggested that the Fiber1/2 subunit vaccine can significantly inhibit liver damage caused by FAdV-4 infection and is more effective in blocking the pathogenicity of FAdV-4 in target organs. In addition, the Fiber1/2 knob subunit vaccine further reduced the viral load in different tissues and virus shedding in chickens than the Fiber2 subunit vaccine. Overall, the Fiber1/2 knob subunit vaccine was more effective than the Fiber2 subunit vaccine. These findings lay the foundation for the development of more effective FAdV-4 subunit vaccines.

Effect of intestinal microbiota on duck short-beak and dwarf syndrome caused by novel goose parvovirus.

Short-beak and dwarf syndrome (SBDS) is caused by infection with novel goose parvovirus (NGPV), which leads to intestinal dysbiosis, developmental delay, short beak, lameness, and paralysis in ducks and is the cause of skeletal health problems. NGPV infection can cause intestinal microbial disturbances, but it is still unclear whether the intestinal microbiota affects the pathogenicity of NGPV. Here, the effects of intestinal microbiota on NGPV-induced SBDS in Cherry Valley ducks were assessed by establishing a duck model for gut microflora depletion/reestablishment through antibiotics (ABX) treatment/fecal microbiota transplanted (FMT). By measuring body weight, beak length, beak width and tarsal length, we found that SBDS clinical symptoms were alleviated in ducks treated with ABX, but not in FMT ducks. Next, we conducted a comprehensive analysis of bone metabolism, gut barrier integrity, and inflammation levels using quantitative real-time PCR (qPCR), enzyme linked immunosorbent assay (ELISA), biochemical analysis and histological analysis. The results showed that ABX treatment improved bone quality reduced bone resorption, mitigated tissue lesions, protected intestinal barrier integrity, and inhibited systemic inflammation in NGPV-infected ducks. Moreover, cecal microflora composition and short-chain fatty acids (SCFAs) production were examined by bacterial 16S rRNA sequencing and gas chromatography. The results revealed that ABX treatment mitigated the decreased abundance of Firmicutes and Bacteroidota in NGPV-infected ducks, as well as increased SCFAs production. Furthermore, ABX treatment reduced the mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) and nuclear factor κB (NF-κB) expression, which are correlated with systemic inflammation in SBDS ducks. These findings suggested that intestinal microflora depletion alleviated NGPV-induced SBDS by maintaining intestinal homeostasis, inhibiting inflammatory response and alleviating bone resorption. These results provide evidence for the pivotal role of intestinal microbiota in the process of SBDS and contribute a theoretical basis for the feasibility of microecological preparation as a method to control SBDS.

When DNA-damage responses meet innate and adaptive immunity.

When cells proliferate, stress on DNA replication or exposure to endogenous or external insults frequently results in DNA damage. DNA-Damage Response (DDR) networks are complex signaling pathways used by multicellular organisms to prevent DNA damage. Depending on the type of broken DNA, the various pathways, Base-Excision Repair (BER), Nucleotide Excision Repair (NER), Mismatch Repair (MMR), Homologous Recombination (HR), Non-Homologous End-Joining (NHEJ), Interstrand Crosslink (ICL) repair, and other direct repair pathways, can be activated separately or in combination to repair DNA damage. To preserve homeostasis, innate and adaptive immune responses are effective defenses against endogenous mutation or invasion by external pathogens. It is interesting to note that new research keeps showing how closely DDR components and the immune system are related. DDR and immunological response are linked by immune effectors such as the cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway. These effectors act as sensors of DNA damage-caused immune response. Furthermore, DDR components themselves function in immune responses to trigger the generation of inflammatory cytokines in a cascade or even trigger programmed cell death. Defective DDR components are known to disrupt genomic stability and compromise immunological responses, aggravating immune imbalance and leading to serious diseases such as cancer and autoimmune disorders. This study examines the most recent developments in the interaction between DDR elements and immunological responses. The DDR network's immune modulators' dual roles may offer new perspectives on treating infectious disorders linked to DNA damage, including cancer, and on the development of target immunotherapy.

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope 110DLDGAW115 was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.

Emerging roles of biological m6A proteins in regulating virus infection: A review.

N6-methyladenosine (m6A) is the most prevalent chemical modifications of intracellular RNA, which recently emerging as a multifaceted effector of viral genomic RNA. As a dynamic process, three groups of biological proteins control the levels of m6A modification in eukaryocyte, designed as m6A writers, erasers, and readers. The m6A writers comprising of methyltransferases complex initiate the modification process. On the contrary, the m6A erasers ALKBH5 or FTO abolish the modification through three-step demethylation: m6A to N6-hydroxymethyl adenosine (hm6A), then hm6A to N6-methyladenosine (f6A), and finally f6A to adenosine. The known m6A readers include the YTH family and the hnRNP family. As m6A modification regulates RNA nuclear exportation, stability, and translation, m6A proteins commonly participate in virus infection by regulating viral genomic RNA synthesis. Moreover, m6A proteins establish molecular linkages between virus genome/viral encode proteins and host cells proteins via their multifunctional roles in cellular RNA metabolism. The m6A writers and erasers directly impact interferon expression and macrophage innate immune responses, facilitating them to act as anti-/pro-viral factors. The m6A readers enable to alter cell metabolism and stress granules (SGs) production to regulate virus-host interactions. Here, the latest progress of m6A proteins in regulating viral infection is reviewed. Demonstrating the roles of m6A proteins will enhance the understanding of epigenetic regulation of virus infection and stimulate the development of novel antiviral strategies.

Recombinant porcine Interferon-α and Interleukin-2 fusion protein (rPoIFNα+IL-2) shows potent anti-pseudorabies virus activity in vitro and in vivo.

Pseudorabies virus (PRV) variants have been widely prevalent since 2011, leading to substantial losses to the swine industry. Although PRV can cause cross-species transmission and induce human infection, no drugs can currently prevent PRV infection. Interferons (IFNs) and interleukin-2 (IL-2) are important cytokines that mediate several biological functions including antiviral activity and immune regulation. In this study, we expressed and purified a recombinant porcine IFN-α and IL-2 fusion protein (rPoIFNα+IL-2), which did not show a cytotoxic effect on PK-15 cells. The antiviral activity was evaluated in PK-15 cells using the cytopathic effect inhibition method, and the results indicated that rPoIFNα+IL-2 can inhibit the replication of PRV, with an antiviral activity of approximately 104 U/mL. Moreover, the proliferation of peripheral blood mononuclear cells was enhanced by rPoIFNα+IL-2. Additionally, rPoIFNα+IL-2 substantially increased the expression of IFN-stimulated genes, including IFIT1, ISG15, MX1, and OAS, which are critical for antiviral activity. Furthermore, rPoIFNα+IL-2 alleviated the clinical symptoms and reduced mortality in mice infected with PRV. Simultaneously, rPoIFNα+IL-2 increased the expression levels of IFN-γ and IL-10 and inhibited the expression of IL-1ß and IL-6. Additionally, the viral DNA copies in different tissues in the rPoIFNα+IL-2-treated group were lower than those in the untreated group. These findings indicate that rPoIFNα+IL-2 may serve as an antiviral agent for the prevention and treatment of PRV infection and may expand the potential function of IFN antiviral drugs in the future.

2B and 3C Proteins of Senecavirus A Antagonize the Antiviral Activity of DDX21 via the Caspase-Dependent Degradation of DDX21.

Senecavirus A (SVA), also known as Seneca Valley virus, is a recently discovered picornavirus that can cause swine vesicular disease, posing a great threat to the global swine industry. It can replicate efficiently in cells, but the molecular mechanism remains poorly understood. This study determined the host's differentially expressed proteins (DEPs) during SVA infection using dimethyl labeling based on quantitative proteomics. Among the DE proteins, DDX21, a member of the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase (DDX) family, was downregulated and demonstrated inhibiting SVA replication by overexpression and knockdown experiment. To antagonize this antiviral effect of DDX21, SVA infection induces the degradation of DDX21 by 2B and 3C proteins. The Co-IP results showed that 2B and 3C did not interact with DDX21, suggesting that the degradation of DDX21 did not depend on their interaction. Moreover, the 3C protein protease activity was necessary for the degradation of DDX21. Furthermore, our study revealed that the degradation of DDX21 by 2B and 3C proteins of SVA was achieved through the caspase pathway. These findings suggest that DDX21 was an effective antiviral factor for suppressing SVA infection and that SVA antagonized its antiviral effect by degrading DDX21, which will be useful to guide further studies into the mechanism of mutual regulation between SVA and the host.

Epidemiological Investigation of Porcine Pseudorabies Virus in Hebei Province, China, 2017-2018.

Pseudorabies (PR) is a serious disease affecting the pig industry in China, and it is very important to understand the epidemiology of pseudorabies virus (PRV). In the present study, 693 clinical samples were collected from Bartha-K61 vaccinated pigs with symptoms of suspected PRV infection between January 2017 and December 2018. All cases were referred for full clinical autopsy with detailed examination of histopathological examination, virus isolation and genetic evolution analysis of the PRV glycoprotein E (gE) gene. In addition, PRV gE antibodies in 3,449 serum samples were detected by the enzyme-linked immunosorbent assay (ELISA). The clinical data revealed that abortion and stillbirth are the most frequent appearances in pregnant sows of those cases. Histopathological examination exhibited a variety of pathological lesions, such as lobar pneumonia, hepatitis, lymphadenitis, nephritis, and typical nonsuppurative encephalitis. A total of 248 cases tested positive for the PRV gE gene. 11 PRV variants were isolated and confirmed by gE gene sequencing and phylogenetic analysis. These strains had 97.1%-100.0% nucleotide homology with the PRV reference strains. Notably, the isolated strains were highly homologous and clustered in the same branch as HSD-1/2019, which caused human acute encephalitis. Serological tests showed that the positive rate of PRV gE antibody in the 3449 serum samples collected from the Hebei Province was 46.27%. In conclusion, PRV variant strains Are high prevalence in the Hebei Province, which not only causes huge economic losses to the breeding industry but also potentially poses a threat to public health.

Emergence and genomic analysis of a novel ostrich-origin GPV-related parvovirus in China.

In recent years, ostrich disease characterized by paralysis and diarrhea has been circulating in some regions of China, causing huge economic losses to the ostrich breeding industry. In our study, clinical samples from diseased ostriches were collected, and only parvovirus was detected. The virus distribution analysis by histopathology and quantitative real-time PCR assays indicated that the virus had a wide range of tissue tropisms. The full-length genome of the ostrich parvovirus (OsPV) was sequenced and comprehensively analyzed. Interestingly, the phylogenetic and alignment results indicated that the OsPV and the goose parvovirus (GPV) form a separate branch. In contrast to GPV strains, OsPV showed 2 new 14 nucleotide deletions in the inverted terminal repeat (ITR) region. Furthermore, recombination analysis indicated that OsPV was a recombination strain between the vaccine strain SYG61v and the virulent strain B strain, with the major parent of OsPV as the SYG61v strain and the minor parent as the B strain. The 14 nucleotide deletions in the ITR region as well as recombination may be some of the reasons for the cross-species transmission of parvovirus from goose to ostrich. The above data will contribute to a better understanding of the molecular biology of the novel OsPV and help to develop the vaccine candidate strain.

The Cellular and Viral circRNAs Induced by Fowl Adenovirus Serotype 4 Infection.

Circular RNAs (circRNAs) are a new class of noncoding RNAs that play vital roles in many biological processes. Virus infection induces modifications in cellular circRNA transcriptomes and expresses viral circRNAs. The outbreaks of Hydropericardium-hepatitis syndrome (HHS) caused by fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry worldwide. To investigate the expression of circRNAs during FAdV-4 infection, we performed transcriptome analysis of FAdV-4-infected leghorn male hepatoma (LMH) cells. In total, 19,154 cellular circRNAs and 135 differentially expressed (DE) cellular circRNAs were identified. The characteristics of the DE cellular circRNAs were analyzed and most of them were related to multiple biological processes according to GO and KEGG enrichment analysis. The accuracy of 10 cellular circRNAs were verified by semiquantitative RT-PCR and sequencing. The change trend was consistent with the RNA sequencing results. Moreover, 2014 viral circRNAs were identified and 10 circRNAs were verified by the same methods. Our analysis showed that seven circRNAs with the same 3' terminal and variable 5' terminal regions were located at pTP protein and DNA pol protein of FAdV-4, which may be generated via alternative splicing events. Moreover, the expression level of viral circRNAs was closely related to the replication efficiency of the virus and partial of the viral circRNAs promoted the replication of FAdV-4. Competing endogenous RNA analysis further showed that the effects of cellular and viral circRNAs on host or viral genes may act via miRNAs. Collectively, our findings first indicate that FAdV-4 infection induced the differential expression of cellular circRNAs and FAdV-4 also expressed viral circRNAs, some of which affected FAdV-4 replication. These findings will provide new clues for further understanding FAdV-4 and provide a basis for investigating host-virus interactions.

Discovery and Characterization of an Aberrant Small Form of Glycoprotein I of Herpes Simplex Virus Type I in Cell Culture.

The 380-to-393-amino-acid glycoprotein I (gI) encoded by herpes simplex virus 1 (HSV-1) is a critical mediator for viral cell-to-cell spread and syncytium formation. Here we report a previously unrecognized aberrant form of gI in HSV-1-infected cells. Production of this molecule is independent of cell type and viral strains. It had an unexpected gel migration size of approximately 23 kDa, was packaged into viral particles, and could be coimmunoprecipitated by antibodies to both N and C termini of gI. Deep sequencing failed to detect alternative RNA splicing, and the invitro transcribed full-length mRNA gave rise to the 23 kDa protein in transfected cells. Combined mass spectrometry and antibody probing analyses detected peptide information across different regions of gI, suggesting the possibility of a full-length gI but with abnormal migration behavior. In line with this notion, the HA insertion mutagenesis revealed a stable fold in the gI extracellular region aa.38-196 resistant to denaturing conditions, whereas small deletions within this region failed the antibodies to detect the fast, but not the slow-moving species of gI. It is also intriguing that the structure could be perturbed to some extent by a gBsyn mutation, leading to exposure or shielding of the gI epitopes. Thus, the HSV-1 gI apparently adopts a very stable fold in its natural form, rendering it an unusual biophysical property. Our findings provide novel insight into the biological properties of HSV gI and have important implications in understanding the viral spread and pathogenesis. IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but its behavior during infection has remained poorly defined. Along with the classic 66 kDa product, here we report a previously unrecognized, approximately 23 kDa form of gI. Biochemical and genetics analyses revealed that this molecule represents the full-length form of gI but adopts a stable fold in its extracellular domain that is resistant to denatured conditions, thus contributing to the aberrant migration rate. Our results revealed a novel property of HSV-1 gI and have important implications in understanding viral pathogenesis.

The Emerging Role of RNA Modifications in the Regulation of Antiviral Innate Immunity.

Posttranscriptional modifications have been implicated in regulation of nearly all biological aspects of cellular RNAs, from stability, translation, splicing, nuclear export to localization. Chemical modifications also have been revealed for virus derived RNAs several decades before, along with the potential of their regulatory roles in virus infection. Due to the dynamic changes of RNA modifications during virus infection, illustrating the mechanisms of RNA epigenetic regulations remains a challenge. Nevertheless, many studies have indicated that these RNA epigenetic marks may directly regulate virus infection through antiviral innate immune responses. The present review summarizes the impacts of important epigenetic marks on viral RNAs, including N6-methyladenosine (m6A), 5-methylcytidine (m5C), 2'-O-methylation (2'-O-Methyl), and a few uncanonical nucleotides (A-to-I editing, pseudouridine), on antiviral innate immunity and relevant signaling pathways, while highlighting the significance of antiviral innate immune responses during virus infection.

Construction and Generation of a Recombinant Senecavirus a Stably Expressing the NanoLuc Luciferase for Quantitative Antiviral Assay.

Senecavirus A (SVA), also known as Seneca Valley virus, is a recently emerged picornavirus that can cause swine vesicular disease, posing a great threat to the global swine industry. A recombinant reporter virus (rSVA-Nluc) stably expressing the nanoluciferase (Nluc) gene between SVA 2A and 2B was developed to rapidly detect anti-SVA neutralizing antibodies and establish a high-throughput screen for antiviral agents. This recombinant virus displayed similar growth kinetics as the parental virus and remained stable for more than 10 passages in BHK-21 cells. As a proof-of-concept for its utility for rapid antiviral screening, this reporter virus was used to rapidly quantify anti-SVA neutralizing antibodies in 13 swine sera samples and screen for antiviral agents, including interferons ribavirin and interferon-stimulated genes (ISGs). Subsequently, interfering RNAs targeting different regions of the SVA genome were screened using the reporter virus. This reporter virus (rSVA-Nluc) represents a useful tool for rapid and quantitative screening and evaluation of antivirals against SVA.

Pseudorabies virus encephalitis in humans: a case series study.

Pseudorabies virus (PRV) is known to cause severe encephalitis in juvenile pigs and various non-native hosts; recent evidences suggest that PRV might cause encephalitis in humans. In a multicenter cohort study in China, next-generation sequencing of cerebrospinal fluid (CSF) was performed to detect pathogens in all patients with clinically suspected central nervous system infections. This study involved all the patients whose CSF samples were positive for PRV-DNA; their clinical features were evaluated, and species-specific PCR and serological tests were sequentially applied for validation. Among the 472 patients tested from June 1, 2016, to December 1, 2018, six were positive for PRV-DNA, which were partially validated by PCR and serological tests. Additionally, we retrospectively examined another case with similar clinical and neuroimaging appearance and detected the presence of PRV-DNA. These patients had similar clinical manifestations, including a rapid progression of panencephalitis, and similar neuroimaging features of symmetric lesions in the basal ganglia and bilateral hemispheres. Six of the patients were engaged in occupations connected with swine production. PRV infection should be suspected in patients with rapidly progressive panencephalitis and characteristic neuroimaging features, especially with exposure to swine.

Induction of Rod-Shaped Structures by Herpes Simplex Virus Glycoprotein I.

The envelope glycoprotein I (gI) of herpes simplex virus 1 (HSV-1) is a critical mediator of virus-induced cell-to-cell spread and cell-cell fusion. Here, we report a previously unrecognized property of this molecule. In transfected cells, the HSV-1 gI was discovered to induce rod-shaped structures that were uniform in width but variable in length. Moreover, the gI within these structures was conformationally different from the typical form of gI, as a previously used monoclonal antibody mAb3104 and a newly made peptide antibody to the gI extracellular domain (ECD) (amino acids [aa] 110 to 202) both failed to stain the long rod-shaped structures, suggesting the formation of a higher-order form. Consistent with this observation, we found that gI could self-interact and that the rod-shaped structures failed to recognize glycoprotein E, the well-known binding partner of gI. Further analyses by deletion mutagenesis and construction of chimeric mutants between gI and gD revealed that the gI ECD is the critical determinant, whereas the transmembrane domain served merely as an anchor. The critical amino acids were subsequently mapped to proline residues 184 and 188 within a conserved PXXXP motif. Reverse genetics analyses showed that the ability to induce a rod-shaped structure was not required for viral replication and spread in cell culture but rather correlated positively with the capability of the virus to induce cell fusion in the UL24syn background. Together, this work discovered a novel feature of HSV-1 gI that may have important implications in understanding gI function in viral spread and pathogenesis.IMPORTANCE The HSV-1 gI is required for viral cell-to-cell spread within the host, but the molecular mechanisms of how gI exactly works have remained poorly understood. Here, we report a novel property of this molecule, namely, induction of rod-shaped structures, which appeared to represent a higher-order form of gI. We further mapped the critical residues and showed that the ability of gI to induce rod-shaped structures correlated well with the capability of HSV-1 to induce cell fusion in the UL24syn background, suggesting that the two events may have an intrinsic link. Our results shed light on the biological properties of HSV-1 gI and may have important implications in understanding viral pathogenesis.

Synthesis of a novel platinum(II) complex with 6,7-dichloro-5,8-quinolinedione and the study of its antitumor mechanism in testicular seminoma.

A new platinum(II) complex, [Pt(ClClQ)(DMSO)Cl] (1), utilizing 6,7-dichloro-5,8-quinolinedione (ClClQ) as a ligand, has been synthesized and fully characterized. Single-crystal X-ray diffraction and other spectroscopic and analytical methods revealed that the coordination geometry of Pt(II) in complex 1 can also be described as a four-coordinated square planar geometry. The aim of the study was to explore the in vitro anticancer properties of complex 1. Our studies showed that complex 1 can regulate the viability of testicular seminoma cells in vitro, including cell proliferation and apoptosis. We further observed negative regulation by complex 1 of the expression levels of the key elements in the phosphoinositide-3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK3ß) pathway, including phosphorylated phosphoinositide-3 kinase (p-PI3K), phosphorylated protein kinase B(p-Akt) and phosphorylated glycogen synthase kinase-3ß (p-GSK3ß). Moreover, the negative effect of complex 1 was reversed by LiCl, a GSK3ß-specific inhibitor of the PI3K signaling pathway. Meanwhile, the levels of Bcl2 associated death promoter (Bad), cytochrome c, active-caspase-3 and active-caspase-9 increased significantly. In conclusion, we observed that complex 1 can regulate the viability of testicular seminoma cells through the PI3K/Akt/GSK3ß signaling pathway and the mitochondria-mediated apoptotic pathway in vitro, and thus, complex 1 may have potential for use as a drug in the treatment of testicular germ cell tumors.

ORF1a of highly pathogenic PRRS attenuated vaccine virus plays a key role in neutralizing antibody induction in piglets and virus neutralization in vitro.

BACKGROUND: Currently, porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viral pathogens in swine in most countries, especially China. Two PRRSV attenuated live vaccine strains (HuN4-F112 and CH-1R) are currently widely used in China. Our previous study showed that HuN4-F112, but not CH-1R, induced high anti-nucleocapsid (N) antibody and neutralizing antibody (NA) titers. Additionally, sera from HuN4-F112 inoculated pigs induced low cross neutralization of CH-1R. METHODS: In the present study, 6 chimeric viruses through exchanging 5' untranslated region (UTR) + open reading frame (ORF)1a, ORF1b, and ORF2-7 + 3'UTR between HuN4-F112 and CH-1R were constructed and rescued based on the infectious clones of rHuN4-F112 and rCH-1R. The characteristics of these viruses were investigated in vitro and vivo. RESULTS: All the three fragments, 5'UTR + ORF1a, ORF1b, and ORF2-7 + 3'UTR, could affect the replication efficiencies of rHuN4-F112 and rCH-1R in vitro. Additionally, both 5'UTR + ORF1a and ORF2-7 + 3'UTR affected the anti-N antibody and NA responses targeting rHuN4-F112 and rCH-1R in piglets. CONCLUSIONS: The 5'UTR + ORF1a region of HuN4-F112 played a key role in inducing NAs in piglets. Furthermore, we confirmed for the first time that ORF1a contains a neutralization region. This study provides important information that can be used for further study of the generation of anti-PRRSV NAs.

Characterization of two novel porcine reproductive and respiratory syndrome virus isolates with deletions in the GP2 gene.

Two newly emerged, porcine reproductive and respiratory syndrome virus (PRRSV) strains (Henan-A10 and A11) were isolated from the sera of aborting sows. Interestingly, both of the isolates could replicate in primary porcine alveolar macrophage (PAM) cells but not in MARC-145 cells. A phylogenetic tree based on the complete genome was constructed and the results showed that Henan-A10 and A11 were most closely related to other highly pathogenic PRRSV (HP-PRRSV) strains. However, genomic sequence analysis showed that Henan-A10 and A11 shared only 96.8-97.8% nucleotide identity with the representative HP-PRRSV strain JXA1. Notably, a 10 amino acids deletion in the GP2 endodomain was identified for the first time. A full-length, infectious cDNA clone of HuN4-F112 (attenuated strain from a HP-PRRSV) was used to construct a chimeric clone with the corresponding deletion in GP2. We found that the deletion did not affect viral growth in MARC-145 cells, indicating that the endodomain of PRRSV GP2 may be variable.

Characterization of two newly emerged isolates of porcine reproductive and respiratory syndrome virus from Northeast China in 2013.

A newly emerged porcine reproductive and respiratory syndrome virus (PRRSV) that has caused severe reproductive losses in sows appeared in some regions of China in 2013. To explore the biology of this new PRRSV and understand more fully genetic diversity in PRRSV isolates from China, the complete genome of the two 2013 Chinese isolates, designated HLJA1 and HLJB1, were analyzed. Genomic sequence analysis showed that HLJA1 and HLJB1 shared 88.6-98.3% nucleotide identity with genotype 2 (North American type, NA-type) isolates, but only 61.1% with the genotype 1 (European type, EU-type) isolate of Lelystad virus, indicating that both these isolates belong to the NA-type PRRSV genotype. Phylogenetic analysis showed that the NA-type PRRSV isolates formed three subgroups (1, 2 and 3); representatives of these subgroups are VR-2332, CH-1a and HUN4, respectively. HLJA1 and HLJB1 belong to subgroup 2. Analysis of NSP2 revealed that HLJA1 has a 48-amino acid deletion at positions 473-480 and 482-521, unlike other HP-PRRSV isolates, while HLJB1 has only a 1-amino acid deletion at position 481 compared with CH-1a. Interestingly, HLJA1 replicated in PAM cells but not in MARC-145 cells, whereas HLJB1 replicated in both cell types. The neutralizing antibody titer of pig hyperimmune sera against HUN4 was significantly higher than that of HLJA1 or HLJB1. Additionally, genetic variability in GP5 and GP3 proteins and in the novel ORF5a protein was evident. In addition to elucidating the genetic relationships between PRRSV isolates, our results suggest that Chinese PRRSV will remain a pandemic virus.